Enzymatic and Non-enzymatic Antioxidants Determination in Ascorbate–Glutathione Cycle

The monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) activity was measured using 1 U of ascorbate oxidase, and the oxidation rate of NADH was followed at 340 nm (Rao and Ormrod, 1995). The glutathione reductase (GR, EC 1.6.4.2) activity was measured according to Foyer and Halliwell (1976), as based on the rate of decrease in the absorbance of NADPH at 340 nm. The extraction and assay of reduced (GSH) and oxidized (GSSG) glutathione were performed as described by Rao and Ormrod (1995). The GSH concentration was obtained by subtracting the GSSG concentration from the total concentration. The levels of total ascorbate (AsA+DHA) and AsA were measured according to Law et al. (1983) with minor modification. The absorbance at 525 nm was read to determine reduced AsA according to the standard curve of known AsA concentrations. DHA was calculated as the difference between the total AsA and reduced AsA. L-galactono-1,4-lactone dehydrogenase (GaILDH) activity was determined according to the method of Tabata et al. (2001). γ-GCS was extracted and measured by the method of Shan and Liang (2010).